Changes in the reproductive function and developmental phenotypes in mice following intramuscular injection of an activin betaA-expressing plasmid

<p>Abstract</p> <p>Background</p> <p>The TGF-beta family protein activin has numerous reported activities with some uncertainty in the reproductive axis and development. The precise roles of activin in in vivo system were investigated using a transient gain of function model.</p> <p>Methods</p> <p>To this end, an expression plasmid, pCMV-rAct, with the activin betaA cDNA fused to the cytomegalovirus promoter, was introduced into muscle of the female adult mice by direct injection.</p> <p>Results</p> <p>Activin betaA mRNA was detected in the muscle by RT-PCR and subsequent Southern blot analysis. Activin betaA was also detected, and western blot analysis revealed a relatively high level of serum activin with correspondingly increased FSH. In the pCMV-rAct-injected female mice, estrus stage within the estrous cycle was extended. Moreover, increased numbers of corpora lutea and a thickened granulosa cell layer with a small antrum in tertiary follicles within the ovary were observed. When injected female mice were mated with males of proven fertility, a subset of embryos died in utero, and most of those that survived exhibited increased body weight.</p> <p>Conclusion</p> <p>Taken together, our data reveal that activin betaA can directly influence the estrous cycle, an integral part of the reproduction in female mice and activin betaA can also influence the embryo development as an endocrine fashion.</p

Integrative characterization of germ cell-specific genes from mouse spermatocyte UniGene library

<p>Abstract</p> <p>Background</p> <p>The primary regulator of spermatogenesis, a highly ordered and tightly regulated developmental process, is an intrinsic genetic program involving male germ cell-specific genes.</p> <p>Results</p> <p>We analyzed the mouse spermatocyte UniGene library containing 2155 gene-oriented transcript clusters. We predict that 11% of these genes are testis-specific and systematically identified 24 authentic genes specifically and abundantly expressed in the testis via in <it>silico </it>and <it>in vitro </it>approaches. Northern blot analysis disclosed various transcript characteristics, such as expression level, size and the presence of isoform. Expression analysis revealed developmentally regulated and stage-specific expression patterns in all of the genes. We further analyzed the genes at the protein and cellular levels. Transfection assays performed using GC-2 cells provided information on the cellular characteristics of the gene products. In addition, antibodies were generated against proteins encoded by some of the genes to facilitate their identification and characterization in spermatogenic cells and sperm. Our data suggest that a number of the gene products are implicated in transcriptional regulation, nuclear integrity, sperm structure and motility, and fertilization. In particular, we found for the first time that Mm.333010, predicted to contain a trypsin-like serine protease domain, is a sperm acrosomal protein.</p> <p>Conclusion</p> <p>We identify 24 authentic genes with spermatogenic cell-specific expression, and provide comprehensive information about the genes. Our findings establish a new basis for future investigation into molecular mechanisms underlying male reproduction.</p

Characterization of eight novel proteins with male germ cell-specific expression in mouse

<p>Abstract</p> <p>Background</p> <p>Spermatogenesis and fertilization are highly unique processes. Discovery and characterization of germ cell-specific genes are important for the understanding of these reproductive processes. We investigated eight proteins encoded by novel spermatogenic cell-specific genes previously identified from the mouse round spermatid UniGene library.</p> <p>Methods</p> <p>Polyclonal antibodies were generated against the novel proteins and western blot analysis was performed with various protein samples. Germ cell specificity was investigated using testes from germ cell-less mutant mice. Developmental expression pattern was examined in testicular germ cells, testicular sperm and mature sperm. Subcellular localization was assessed by cell surface biotin labeling and trypsinization. Protein localization and properties in sperm were investigated by separation of head and tail fractions, and extractabilities by a non-ionic detergent and urea.</p> <p>Results</p> <p>The authenticity of the eight novel proteins and their specificity to spermatogenic cells were confirmed. In examining the developmental expression patterns, we found the presence of four proteins only in testicular germ cells, a single protein in testicular germ cells and testicular sperm, and three proteins in the testicular stages and mature sperm from the epididymis. Further analysis of the three proteins present in sperm disclosed that one is located at the surface of the acrosomal region and the other two are associated with cytoskeletal structures in the sperm flagellum. We name the genes for these sperm proteins Shsp1 (Sperm head surface protein 1), Sfap1 (Sperm flagellum associated protein 1) and Sfap2 (Sperm flagellum associated protein 2).</p> <p>Conclusion</p> <p>We analyzed eight novel germ cell-specific proteins, providing new and inclusive information about their developmental and cellular characteristics. Our findings will facilitate future investigation into the biological roles of these novel proteins in spermatogenesis and sperm functions.</p

5-Deoxy-Δ 12,14 -Prostaglandin J 2 Down-Regulates Activin-Induced Activin Receptor, Smad, and Cytokines Expression via Suppression of NF-B and MAPK Signaling in HepG2 Cells

15-Deoxy-Δ 12,14 -prostaglandin J 2 (15d-PGJ 2 ) and activin are implicated in the control of apoptosis, cell proliferation, and inflammation in cells. We examined both the mechanism by which 15d-PGJ 2 regulates the transcription of activin-induced activin receptors (ActR) and Smads in HepG2 cells and the involvement of the nuclear factor-B (NF-B) and mitogen-activated protein kinase (MAPK) pathways in this regulation. Activin A (25 ng/mL) inhibited HepG2 cell proliferation, whereas 15d-PGJ 2 (2 M and 5 M) had no effect. Activin A and 15d-PGJ 2 showed different regulatory effects on ActR and Smad expression, NF-B p65 activity and MEK/ERK phosphorylation, whereas they both decreased IL-6 production and increased IL-8 production. When costimulated with 15d-PGJ 2 and activin, 15d-PGJ 2 inhibited the activin-induced increases in ActR and Smad expression, and decreased activin-induced IL-6 production. However, it increased activin-induced IL-8 production. In addition, 15d-PGJ 2 inhibited activininduced NF-B p65 activity and activin-induced MEK/ERK phosphorylation. These results suggest that 15d-PGJ 2 suppresses activin-induced ActR and Smad expression, down-regulates IL-6 production, and up-regulates IL-8 production via suppression of NF-B and MAPK signaling pathway in HepG2 cells. Regulation of ActR and Smad transcript expression and cytokine production involves NF-B and the MAPK pathway via interaction with 15d-PGJ 2 /activin/Smad signaling

Systematic identification and integrative analysis of novel genes expressed specifically or predominantly in mouse epididymis

BACKGROUND: Maturation of spermatozoa, including development of motility and the ability to fertilize the oocyte, occurs during transit through the microenvironment of the epididymis. Comprehensive understanding of sperm maturation requires identification and characterization of unique genes expressed in the epididymis. RESULTS: We systematically identified 32 novel genes with epididymis-specific or -predominant expression in the mouse epididymis UniGene library, containing 1505 gene-oriented transcript clusters, by in silico and in vitro analyses. The Northern blot analysis revealed various characteristics of the genes at the transcript level, such as expression level, size and the presence of isoform. We found that expression of the half of the genes is regulated by androgens. Further expression analyses demonstrated that the novel genes are region-specific and developmentally regulated. Computational analysis showed that 15 of the genes lack human orthologues, suggesting their implication in male reproduction unique to the mouse. A number of the novel genes are putative epididymal protease inhibitors or β-defensins. We also found that six of the genes have secretory activity, indicating that they may interact with sperm and have functional roles in sperm maturation. CONCLUSION: We identified and characterized 32 novel epididymis-specific or -predominant genes by an integrative approach. Our study is unique in the aspect of systematic identification of novel epididymal genes and should be a firm basis for future investigation into molecular mechanisms underlying sperm maturation in the epididymis

Genomic organization and role of the sperm surface protein, fertilin

The sperm surface protein fertilin is a heterodimer composed of $\alpha$ and $\beta$ subunits that are the first identified members of the ADAM family. The present dissertation describes the chromosomal localization and gene structure of fertilin, and the functional characterization of fertilin $\beta$ using a gene knockout approach.^ Chromosomal locations of four mouse ADAM family genes including fertilin $\alpha$ and fertilin $\beta$ were determined by genetic mapping. These genes have been given the locus symbols Ftna (fertilin $\alpha),$ Ftnb (fertilin $\beta),$ Adam4 (ADAM 4), and Adam5 (ADAM 5). They were mapped to mouse chromosomes, 5, 14, 9 and 8, respectively, revealing a dispersed localization.^ Analysis of the mouse fertilin $\beta$ gene revealed that the gene is present as a single copy, spanning about 55 kb in the genome. The fertilin $\beta$ gene consists of at least 20 exons that average 114 bp in size, interrupted by 19 introns. Comparison of genomic organization between mouse fertilin $\beta$ and the previously sequenced ADAM family gene, human MDC, showed 12 conserved exon-intron boundaries, revealing their evolutionary relationship. Additionally, it was found that more than one gene for fertilin $\alpha$ is present in the mouse genome.^ Mice carrying a targeted mutation in the fertilin $\beta$ gene were generated to test hypotheses that fertilin acts in sperm-egg binding and fusion, and egg activation. Adhesion of sperm lacking fertilin $\beta$ to the egg plasma membrane is dramatically decreased. Fusion of mutant sperm with the egg membrane occurs with only a slight reduction. When mutant sperm fuse with the egg membrane, eggs are activated normally. Furthermore, it was found that mutant sperm are defective in additional steps of fertilization. The progression of mutant sperm from the uterus into the oviduct is severely impaired and mutant sperm fail to adhere to the egg coat, the zona pellucida. Male homozygous mutants are infertile despite normal mating. These results indicate that fertilin is important for multiple sperm functions in fertilization.

Testicular germ cell–specific lncRNA, Teshl , is required for complete expression of Y chromosome genes and a normal offspring sex ratio

International audienceHeat shock factor 2 (HSF2) regulates the transcription of the male-specific region of the mouse Y chromosome long arm (MSYq) multicopy genes only in testes, but the molecular mechanism underlying this tissue specificity remains largely unknown. Here, we report that the testicular germ cell-specific long noncoding RNA (lncRNA), NR_038002, displays a characteristic spatiotemporal expression pattern in the nuclei of round and elongating spermatids. NR_038002-knockout male mice produced sperm with abnormal head morphology and exhibited reduced fertility accompanied by a female-biased sex ratio in offspring. Molecular analyses revealed that NR_038002 interacts with HSF2 and thereby activates expression of the MSYq genes. We designate NR_038002 as testicular germ cell-specific HSF2-interacting lncRNA (Teshl). Together, our study is the first to demonstrate that the testis specificity of HSF2 activity is regulated by the lncRNA Teshl and establishes a Teshl-HSF2-MSYq molecular axis for normal Y-bearing sperm qualities and consequent balanced offspring sex ratio

15-deoxy-Δ12,14-prostaglandin J2 Down-Regulates Activin-Induced Activin Receptor, Smad, and Cytokines Expression via Suppression of NF-κB and MAPK Signaling in HepG2 Cells

15-Deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) and activin are implicated in the control of apoptosis, cell proliferation, and inflammation in cells. We examined both the mechanism by which 15d-PGJ2 regulates the transcription of activin-induced activin receptors (ActR) and Smads in HepG2 cells and the involvement of the nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) pathways in this regulation. Activin A (25 ng/mL) inhibited HepG2 cell proliferation, whereas 15d-PGJ2 (2 μM and 5 μM) had no effect. Activin A and 15d-PGJ2 showed different regulatory effects on ActR and Smad expression, NF-κB p65 activity and MEK/ERK phosphorylation, whereas they both decreased IL-6 production and increased IL-8 production. When co-stimulated with 15d-PGJ2 and activin, 15d-PGJ2 inhibited the activin-induced increases in ActR and Smad expression, and decreased activin-induced IL-6 production. However, it increased activin-induced IL-8 production. In addition, 15d-PGJ2 inhibited activin-induced NF-κB p65 activity and activin-induced MEK/ERK phosphorylation. These results suggest that 15d-PGJ2 suppresses activin-induced ActR and Smad expression, down-regulates IL-6 production, and up-regulates IL-8 production via suppression of NF-κB and MAPK signaling pathway in HepG2 cells. Regulation of ActR and Smad transcript expression and cytokine production involves NF-κB and the MAPK pathway via interaction with 15d-PGJ2/activin/Smad signaling